Mammals require molecular oxygen (O2) for essential metabolic processes, including oxidative phosphorylation in which O2 serves as electron acceptor during ATP formation. Hypoxia occurs when the demand for molecular oxygen exceeds supply. Hypoxia causes systemic, local, and intracellular homeostatic responses that include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann (1992) Physiol. Rev. 72:449–489), neovascularization in ischemic myocardium (White et al. (1992) Circ. Res. 71:1490–1500), and glycolysis in cells cultured at reduced O2 tension (Wolfle et al. (1983) Eur. J. Biochem. 135:405–412). These adaptive responses either increase O2 delivery or activate alternate metabolic pathways that do not require O2.
Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza (1994) Hematol. Oncol. Clinics N. Amer. 8:863–884), vascular endothelial growth factor (Shweiki et al. (1992) Nature 359:843–845; Banai et al. (1994) Cardiovasc. Res. 28:1176–1179; Goldberg & Schneider (1994) J. Biol. Chem. 269:4355–4359), and glycolytic enzymes (Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496–6500; Semenza et al. (1994) J. Biol. Chem. 269:23757–23763).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and therefore blood O2-carrying capacity (Jelkmann (1992) supra; Semenza (1994) supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3′-flanking region. A trans-acting factor that binds to this transcriptional activation region has been identified: hypoxia-inducible factor 1α (HIF-1α). Several lines of evidence indicate that HIF-1α is a physiological regulator of EPO transcription. Inducers of EPO expression, including 1% O2, cobalt chloride, and desferrioxamine, induce HIF-1α DNA binding activity with similar kinetics. Moreover, inhibitors of EPO expression, including actinomycin D, cycloheximide, and 2-aminopurine, blocked induction of HIF-1α activity. Mutations in the EPO 3′-flanking region eliminate HIF-1α binding and HIF-1α transcriptional activation (Semenza (1994) supra).
Induction of HIF-1 activity by 1% O2, CoCl2, or desferrioxamine (DFX) has been detected in many mammalian cell lines (Wang & Semenza (1993a) Proc. Natl. Acad. Sci. USA 90:4304–4308). Reporter genes linked to the EPO enhancer and transfected into non-EPO-producing cells were actively transcribed by hypoxia-inducible factor (Wang & Semenza (1993a) supra; Maxwell et al. (1993) Proc. Natl. Acad. Sci. USA 90:2423–2427). RNAs encoding several glycolytic enzymes were induced by 1% O2, CoCl2, or DFX in EPO-producing Hep3B or non-producing HeLa cells. However, cycloheximide blocked such induction. Moreover, glycolytic gene sequences containing HIF-1 binding sites exhibited hypoxia-inducible transcription in transfection assays (Firth et al. (1994) supra; Semenza et al. (1994) supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.
Angiogenesis, or the process of producing new blood vessels in the body, is a key step in a number of biological responses to injury, stroke, or sudden loss of oxygen. In most cells this process is under tight control by a series of oxygen-sensitive proteins that act in concert to prevent undue blood vessel formation. A key protein mediator of this response is vascular endothelial growth factor (VEGF), a potent stimulator of blood vessel growth. Tumor cells often express this protein at levels 3–10 times higher than normal cells. Consequently, much attention has been directed at developing anticancer strategies focused on inhibiting the action of VEGF.
However, despite its positive role in healing injured tissues and in responding to hypoxia, little or no attention has been placed on stimulating the body to produce more VEGF. Instead, most research has been targeted on providing anti-angiogenic approaches for use in cancer treatment. There are a number of diseases, conditions and injuries that would benefit from VEGF activation. Accordingly, a need exists for factors that can stimulate VEGF production.